Hypothalamic control of prolactin secretion was studied in ovariectomized ewes by comparing the effects of hypothalamo-pituitary disconnection (HPD) and sham-operation (sham-HPD). HPD caused a two-fold increase in plasma prolactin concentrations on days 1 and 7 following surgery during anoestrus and a tenfold increase during the breeding season. Thereafter, concentrations gradually declined to be similar to those in sham-HPD ewes by day 43 (breeding season) and day 145 (anoestrus). The maximum plasma prolactin response to HPD was similar during the two seasons (anoestrus: 128 ± 15 vs breeding season: 118 ± 13 μg/l). Sham-HPD had no effect on plasma prolactin concentrations. Prolactin pulse frequency was not affected by HPD, but increases in plasma prolactin concentrations were associated with increases in pulse amplitude. At the time of the normal anoestrus, plasma prolactin concentrations rose in both the HPD and sham-HPD ewes, raising the question of extra-hypothalamic regulation of seasonal changes in prolactin secretion. Plasma LH and FSH concentrations became undetectable in HPD ewes but were unaltered in sham-HPD ewes. We conclude that hypothalamic inhibition of pituitary prolactin secretion in the sheep can be demonstrated by HPD but that this effect is not sustained. This transience may indicate that additional requirement of hypothalamic-releasing factors in the control of prolactin release. In addition, the surgically isolated ovine pituitary of the HPD animal has an inherent pulsatile secretion of prolactin.